Posted: February 1st, 2023

Principles and steps involved in cDNA synthesis

Since RNA is the starting material for cDNA formation, firstly RNA is extracted by lysing of host cells followed by purification using phenol-chloroform, bead based extraction and silica column based methods. These systems help in the separation of RNA from lipids, phenolic compounds and carbohydrates.

Further RNA sample is subjected to DNAses and proteinase K treatments which digest undesired DNA and proteins in the sample.

In addition, RNA integrity is preserved using RNAses and chaotropic reagents such as guanidium isothiocynate, sodium dodecyl sulphate, phenol and chloroform.

Finally total and purified RNA is extracted using a gradient of alcohol precipitations.

This is followed by reverse transcription step which is achieved in two steps; First strand and second strand cDNA synthesis.

In the first strand synthesis, enriched mRNA is reverse transcribed using reverse transcriptase enzyme, oligo dT primers, random hexamers or gene specific primers to convert into cDNA. The primers in presence of enzyme bind to poly A tail, random regions or to a specific gene respectively and begin the synthesis of cDNA. The new strand formed from the mRNA is termed as first strand of cDNA and they exist as RNA-DNA hybrids.

In the second strand synthesis, the hybrid is displaced by cDNAs using multiple second strand synthesis methods. Here the RNAse H activity of reverse transcriptase cleaves the hybrid and degrades the mRNA. This is followed by second strand cDNA synthesis using DNA polymerase. Multiple rounds of second strands are synthesized and these steps are performed in a thermocycler using polymerase chain reaction principle as each of the enzymes act at their own optimal temperatures.

Characteristics
Complementary DNA is defined as the DNA synthesized from single stranded RNA.

It is commonly referred to as cDNA, formed from mRNA as the template.

The synthesis of cDNA is performed in vitro using reverse transcription reaction.

It involves principles of molecular biology and recombinant DNA technology.

cDNAs are importantly used to clone eukaryotic genes into a prokaryotic system, as they possess enormous and rapid replication ability.

The mRNAs of eukaryotic cells are extracted, reverse transcribed in vitro into cDNAs and are transformed into the recipient cell.

cDNAs are also naturally produced by retroviruses such as HIV and Simian immunodeficiency viruses. In this case their single stranded RNAs are transformed into DNA (cDNA) using viral retroviruses and then are integrated into the host genome to form a provirus.

cDNA terminology is often used in bioinformatics context to refer to mRNA transcript sequences that are expressed as DNA bases rather than RNA bases.

They have huge applications in the field of gene expression studies.